This project will evaluate mechanisms for generation of partially reduced oxygen species (ROS) by pulmonary endothelium during lung ischemia. The studies are currently supported by an R-01 grant which will be relinquished if the SCOR is funded. Our primary experimental model is the isolated, perfused rat lung that is continuously ventilated in order to maintain tissue oxygenation and ATP during global ischemia +/- reperfusion. Additional studies utilize cultured endothelial cell preparations. During our present period of grant support, we have demonstrated generation of ROS and oxidative injury to lung that occurs during the ischemic period and have provided evidence for a new mechanism of ROS production, i.e., ROS production in association with endothelial cell membrane depolarization. ROS generation also was demonstrated in cultured bovine pulmonary artery endothelial cells and in the perfused lung in the presence of membrane depolarizing solutions (high K+) and with anoxia/reoxygenation produced by ventilating lungs with N2 and then 02. Studies with metabolic inhibitors indicated that ROS production with lung ischemia and with anoxia/reoxygenation occurred through different pathways and indicated a role for NADPH oxidase in lung ischemic injury. The specific aims of the present proposal are to: 1) evaluate endothelial generation of ROS through the use of fluorescence imaging in situ and to determine the temporal sequence between endothelial depolarization, changes in intracellular Ca++ and FE2+/3+ and ROS generation; 2) develop an artificial capillary system for the in vitro evaluation of the role of shear stress and shear stress adaptation on endothelial ROS generation with ischemia; and 3) evaluate the role of the NADPH oxidase pathway as the source of ROS and endothelium using inhibitors, a knock-out mouse model, and antisense technology. This project will provide new information concerning a mechanism for initiation of lung oxidative injury and will document the presence of NADPH oxidase in endothelium and a possible pathophysiologic role in lung ischemia injury. This mechanism is of potential importance in ARDS as a source of ROS generation in association with focal capillary obstruction.